thermomycolin

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed in compliance with GLP and according procedures similar to OECD TG 401

Remarks:

/ The acclimatization period was 4 days. One female rat at the lowest dose level was excluded due to misdosing.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

24 Feb 1987

Deviations:

yes

Remarks:

/ The acclimatization period was 4 days. One female rat at the lowest dose level was excluded due to misdosing.

GLP compliance:

yes

Test type:

other:

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Subtilisin

EC Number:

232-752-2

EC Name:

Subtilisin

Cas Number:

9014-01-1

Molecular formula:

Not applicable, please see remarks.

IUPAC Name:

Subtilisin

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Møllegaards Breeding Center Ltd., Ll. Skensved, Denmark
- Fasting period before dosing: 18 hours
- Housing: 5 animals per cage, Macrolon type IV, separate sex
- Weight at time of dosing: between 80 - 104 g
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: 4 days
- Temperature (°C): 17-26C
- Humidity : 31-55 %

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Tap water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL, corresponding to 0, 29, 58 and 116 mg TOS/mL
- Amount of vehicle (if gavage): constant volume 20 mL/kg b.w.
- Justification for choice of vehicle: The test material is water soluble and any human exposure will be in aqueous solutions.
- Purity: tap water

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg

Doses:

0, 1000, 2000 and 4000 mg/kg bw, corresponding to 0, 580 mg, 1160 mg and 2320 mg TOS/kg body weight

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: 30 min., 2 hrs and daily after dosing. Weighing: once weekly
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

LD50 was determined by iterative probit method from log-dose response (Finney DJ. Probit Analysis. Cambridge University Press, 1971).

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 800 mg/kg bw

Based on:

test mat.

95% CL:

> 1 200 - < 2 300

Mortality:

All animals in the top dose group died within 2 hours after dosing. In the mid dose group, four males and three females died within five hours after dosing and one female rat died 23 hours after dosing. See also table below

Clinical signs:

Affected animals showed decreased activity, head drop and diarrhoea. No clinical signs in low dose group and negative controls.

Body weight:

No difference in body weights and body weight gains between the negative control group and the treated groups (surviving animals).

Gross pathology:

The animals that died shortly after dosing all showed extensive gastrointestinal bleedings, some also bleedings from the nostrils and anus. Surviving animals sacrificed at day 15 showed no abnormalities.

Other findings:

- Potential target organs: No dose related organ changes were found in the surviving animals.

Any other information on results incl. tables

Table showing the mortality

Dosage mg/kg	Group size		Mortality %
	Males	Females	Males	Females
4000 2000 1000 0	5 5 5 5	5 5 4* 5	100 80 0 0	100 80 0 0

* One female excluded due to misdosing

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 value was 1.8 g/kg. The main clinical symptoms and the causes of death were ascribed to gastrointestinal disturbances. From other studies, it is known that when the proteolytic activity of Subtilisin is inactivated by treatment with hydrochloric acid, the toxicological potential is decreased significantly. Thus, the proteolytic activity contributes essentially to the toxic effect (Please see the HERA document on subtilisin for further detail).

CLP: Acute Oral 4, H302

Executive summary:

The acute toxicity of Subtilisin, batch PPA 1619, was investigated according to the principles of the later OECD test guideline 401. Four groups of five male and five female Wistar rats received the test material at a dosage of 0, 1000, 2000 and 4000 mg test material per kg body weight by oral administration (gavage). The animals were subjected to clinical observations daily for a fourteen-day observation period. Gross necropsy was carried out on all rats that died during the study or were sacrificed at termination of the study. All animals in the top dose group died within 2 hours after dosing. In the mid dose group, four males and four females died within 5 - 23 hours after dosing. Main clinical signs were decreased activity, head drop and diarrhoea. All decedents showed extensive gastrointestinal bleedings, some also bleedings from the nostrils and anus. The low dose group and the negative controls showed no clinical signs. All surviving animals had normal body weights and body weight gains. Surviving animals sacrificed at day 15 showed no abnormalities at necropsy. The oral LD50 value was determined to be 1800 mg/kg body weight for both male and female rats.